The proposed studies are aimed at understanding how the HIV-1 Gag polyprotein and its constituent matrix (MA) and capsid (CA] domains function during viral replication. During the current funding period we identified the structural determinants responsible for targeting HIV-1 Gag to virus assembly sites on the plasma membrane (PM). Speciflcally, we showed that phosphatidyIinositoI-4,5-bisphosphate (PIP2), a constituent ofthe PM, binds to a conserved cleft on the HIV-1 MA protein and triggers exposure ofthe N-terminal myristyl group. The unsaturated 2'-acyl chain of PIP2 is sequestered within the cleft ofthe protein, and exposure ofthe saturated 1'- chain and myristyl group explains how HIV-1 Gag is targeted specifically to lipid raft assembly sites. We further showed that conservative mutations that mistarget Gag to the cytoplasm in vivo turn off the HIV-1 myristyl switch; that HlV-2 Gag trafficking is also P1P2 dependent; and that the switch behavior of HIV-2 MA is more tightly regulated than that of HIV-1, which could explain the poor replication properties ofthe virus in some cell types and in humans. In addition, we determined the first atomic level strucuture of an antiviral assembly inhibitor bound to the HIV-1 CA protein. We are now poised to extend studies to macromolecular interacdons that occur during HIV assembly, including interactions among viral constituents and between viral and cellular constituents. A new system for detecting interactions between HIV-1 MA and the intra-viral segment ofthe envelope glycoprotein GP41 has been developed, which will be used to identify specific interactions and structures responsible for recuriting the envelope protein into assembling particles. Studies ofthe myristyl switch will be extended to animal viruses commonly used as models for HIV-1 and for drug testing, Including SIV and BLV. New efforts will be made to identify compounds that bind to conserved sites on HlV-1, HIV-2, and SIV MA and inhibit myristate exposure. Finally, based on our high resolution structure ofthe CA:CAP-1 complex, and as part ofa new collaboration with Vironova Pharmaceuticals (Sweden), we intend to rationally design capsid assembly inhibitors with improved antiviral properties.